自噬通过选择性降解Wwp2促进肺癌细胞“干性化”致AZD9291耐药的机制研究

The third-generation EGFR-TKI AZD9291 is an effective drug for curbing the resistance problem of first-generation EGFR-TKI, but it still faces the problem of resistance. It was reported that autophagy promoted the acquired resistance of lung cancer cells to tyrosine kinase inhibitors, however, the mechanisms underlying it are elusive. Here our study found that the stemness of AZD9291-resistant tumor cells elevated, including the expression of pluripotent protein Sox2 and Oct4. Wwp2 is the E3 ligase responsible for the ubiquitinproteasome pathway degradation of Sox2 and Oct4. The selective degradation of Wwp2 by autophagy increases the stability of Sox2 and Oct4 and helps maintain the stemness. Thus, we speculated that the protective autophagy played an important role in maintaining stemness of tumor cells by selectively degrading Wwp2 and enhancing the stability of Sox2 and Oct4. In this proposal, we will further extend our study to determine the regulatory mechanism of autophagy on maintaining stemness, demonstrate the molecular mechanism of the autophagic selective degradation of Wwp2, in order to provide new strategies to overcome the problem of AZD9291 resistance.

三代EGFR-TKI AZD9291是一代EGFR-TKI耐药后的有效药物，但其同样面临耐药困扰。研究发现，保护性自噬是肺癌细胞TKI获得性耐药的原因之一，但是自噬致TKI耐药的机制不明。我们前期工作发现，AZD9291耐药后肿瘤干性明显增强，干细胞关键基因Sox2、Oct4表达在AZD9291耐药细胞中明显升高。自噬选择性降解Sox2和Oct4的E3泛素连接酶Wwp2从而抑制Sox2和Oct4的蛋白酶体降解途径，因此，我们推测AZD9291耐药后发生的保护性自噬通过促进Wwp2降解抑制Sox2、Oct4降解从而增强肿瘤细胞干性。本课题拟研究AZD9291耐药前后自噬及肿瘤干性的变化、自噬选择性降解Wwp2对Sox2、Oct4在AZD9291耐药中的机制，寻求克服AZD9291耐药的新策略。

三代EGFR-TKI AZD9291是一代EGFR-TKI耐药后的有效药物，但其同样面临耐药困扰。研究发现，保护性自噬是肺癌细胞TKI获得性耐药的原因之一，但是自噬致TKI耐药的机制不明。本课题重点探讨了自噬通过调控肿瘤细胞干性致AZD9291耐药的分子机制，以期阐明自噬对肿瘤干性的调控作用及其在AZD9291耐药中的作用。重要结果及关键数据如下：1）发现自噬增强是除了EGFR,ALK,MET等基因突变外，一种广泛存在于AZD9291耐药患者中的耐药机制。2）阐明自噬通过增强肿瘤干细胞干性促进AZD9291耐药。抑制自噬之后，AZD9291耐药细胞中的干细胞样的细胞群明显减少，细胞对AZD9291的敏感性增强。3）阐明Sox2和ALDH的表达对肺癌干细胞干性维持及对AZD9291耐药的重要作用。4）发现参与自噬的beclin1基因可增强Sox2和ALDH蛋白稳定性，协助维持肿瘤干细胞干性特征，而atg5基因并无此作用。5）阐明自噬抑制剂可提高AZD9291的敏感性。因此，我们得出结论，beclin1依赖的自噬途径通过增强肿瘤干细胞干性进而促进AZD9291耐药，抑制自噬可增强肺癌细胞对AZD9291敏感性。我们的研究不仅揭示了一种beclin1依赖，非atg5依赖的自噬在AZD9291耐药中的作用，也为AZD9291的临床应用提供了理论支持和基础依据。另外受该基金资助，我们发现自噬通过选择性自噬降解HK2蛋白调控糖酵解，继而导致抑制自噬会促进糖酵解的增强。